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The monoliths were created by an unseen alien race, known only as the "Firstborn." The first monolith to be discovered was officially dubbed Tycho Magnetic Anomaly One, or TMA-1. Three of the other monoliths were also given "TMA" designations, although none of them except the Tycho Monolith were located on the moon. TMA-1 was also the only monolith that emitted a magnetic field, so the designation is something of a double misnomer.

The monoliths are capable of many different functions. TMA-0 (the retroactively numbered monolith discovered in Africa) and the Europa Monolith served as a catalyst for evolution, encouraging the development of sapience in lifeforms. TMA-1 was a sort of "burglar alarm," alerting the aliens that the humans had left Earth. TMA-1 reported to its "brother," TMA-2, found in orbit around Jupiter. Humans were able to identify the signal sent from TMA-1 towards TMA-2, alerting the humans to the existence of TMA-2. This was the catalyst for the Discovery One and later, the joint US-Soviet Leonov missions. While the monoliths remain mostly a mystery to humans, it is hypothesized that the magnetic anomaly on the Moon, and the signal sent upon the Sun touching TMA-1 for the first time after its excavation, were purposely done by the unseen alien race to compel humans to explore farther and encourage space travel. The Jupiter monolith served as a gateway to a transit system, opening up when a decided specimen was found; in one case it was David Bowman, who was pulled into the monolith and was turned into the "Star Child," an entity of pure energy who functioned as a probe for the Firstborn.

The monoliths could also replicate themselves, generating a cloud, or rather a giant disc, made up of millions of monoliths. One such assemblage was formed to increase the mass of Jupiter, inducing nuclear fusion and igniting the gas giant to create Lucifer, all to facilitate evolution on Europa. Two more clouds were formed 1,000 years later—one to block light from the sun to Earth and the other to block light from Lucifer to the human bases on Ganymede—to cause the extinction of the human race, because they posed a threat to the Europans. Because of this, the humans unleashed a destructive computer virus series on the monoliths, which were destroyed.

All the monoliths, though they vary greatly in size, were fashioned to the exact proportions of 1:4:9, the squares of the integers 1, 2, and 3. This precise measurement ruled out any naturally occurring phenomena, and because the first of the monoliths was placed more than 3 million years ago, humans could not possibly have made them. Upon entering the monolith, David Bowman discovered that the ratio of proportions continued in a similar ratio in additional dimensions, which were unknown to humans at the time.

The Africus monolith, a “New Rock”, was met by a tribe of man-apes, 3 mya, in Africa on Earth. By mysterious means, the monolith probed the man-ape's minds, carving the path to sapience. It served as a catalyst for intelligence seen in modern humans.

The Africus monolith was the third monolith discovered by modern man, though it was first met by the potential intelligence of the man-ape. Thus, it was designated as TMA-0.

In 3001, newly revived Frank Poole finds out that the remains of this monolith have been unearthed. It was presumably abandoned by the Firstborn after its mission was complete as it was not protected like the other monoliths were. The remains were destroyed after the computer virus was unleashed.

The Tycho Monolith, designated TMA-1, was first discovered by humans on Earth's moon in 1999. TMA-1 was discovered when a low-altitude magnetic survey was done on the Moon, and an anomaly was shown in the data: an extremely intense magnetic field in the crater Tycho. At first, scientists thought it might be an outcrop of magnetic rock, but all the evidence was against it. Not even a giant nickel–iron meteorite could produce a field as intense as this. The source of the anomaly was found buried 40 feet below the lunar surface.

During Dr. Heywood Floyd's inspection of the TMA-1, the sun rose over Tycho crater, and for the first time in over 4 million years, sunlight hit the monolith, unleashing a powerful radio blast aimed directly into the Jovian zone—specifically ar the Jovian Monolith. After the radio-blast, the monolith remained inert and its magnetic field had disappeared.

The Tycho Monolith was later transferred to Earth and placed in front of the United Nations building in New York City.

TMA-1 was destroyed along with the other monoliths when “the virus” was released on them shortly after the start of the fourth millennium.

Jovian Monolith, designated TMA-2, was the second monolith discovered. It was found by Commander David Bowman of the Discovery spacecraft. TMA-2 was found at the Lagrange point between Io and Jupiter, and was commonly called the Jupiter Monolith. The Jovian monolith measured nearly 2 kilometers long.

After Discovery reached Jupiter, Bowman parked the spacecraft at the Lagrange point between Io and Jupiter, since scientists back on Earth had pinpointed this to be the destination of the radio signal. Upon arrival, Bowman went out in a pod to get a closer look at the huge monolith. As he got closer, he saw that "the thing's hollow—it goes on forever—and, oh my God, It's full of stars!"

Bowman was pulled into the monolith, going on a fantastical, almost surreal journey through time and space. In the end, Dave was turned into the Star Child, an immortal being of pure energy, capable of doing whatever he wanted, while obeying the orders from the monoliths and the Firstborn.

When the Cosmonaut Alexei Leonov arrived at Jupiter, another strange chain of events occurred, ultimately transforming Jupiter into the minisun, Lucifer. The Jupiter monolith vanished near the end of the Leonov mission. It was soon discovered that it had gone to Jupiter and replicated itself millions of times over, transforming the planet into a sun. These millions of monoliths were presumably destroyed when the planet was converted.

The Monolith Gather comes to collect a Monolith that has fused with Aaron Stack, a.k.a. the Machine Man.

In 2001: A Space Odyssey issue #8 (July 1977), a monolith gave sentience to a robot named X-51 (also known as "Mister Machine"). Before encountering the monolith, he is the only survivor of a series of robots, and raised as a human son of scientist Abel Stack, who was killed removing his auto-destruct mechanism. He eventually spun off his own title, Machine Man, who integrated him into the Marvel Universe. References from 2001 were mostly ignored, but in the final issue it was revealed that the creators of the monolith were the Celestials, who had sent it to help record vast amounts of data over the centuries.